Japanese Patent Public Disclosure No. 72601/2000: This application was filed with the Japanese Patent Office on Aug. 31, 1998 as Japanese Patent Application No. 245052/1998. The title of the invention was xe2x80x9ca method for preserving extracted mammalian organsxe2x80x9d and the inventor was Kunihiro Seki, D. Sc., the same as the inventor of the present invention. The application was laid open to public inspection in Japan on Mar. 7, 2000 and is incorporated herein by reference to the specification and the drawings.
1. Technical Field of the Invention
The present invention relates to a method for prolonged storage of extracted mammalian organs and such organs that have been preserved for use in transplants.
2. Prior Art
Clinical transplants of human organs such as lungs, heart, liver, kidneys and pancreas are routinely performed today. However, as the number of patients waiting for organ transplants increases yearly, the shortage of donors has become a serious problem and the waiting time to surgery is also increasing. Even if a donor is found, his or her organs cannot be effectively used in transplants since nothing like blood banks exist for organs that are fully equipped with the ability to preserve organs for prolonged periods and allow for efficient supply of organs.
Organs to be transplanted are most commonly preserved by cold storage but the preservation limit is about 4-24 hours (Cooper J D, Patterson G A, Trulock E P et al.; J. Thorac. Cardiovasc. Surg. 107, 460-471, 1994). In experiments using University of Wisconsin Solution (UWS) as a medium for cold storage of hearts from rats, rabbits and baboons before resuscitation, the time limit was 6-18 hours (Makowka I, Zerbe T R, Champman F et al.; Transplant Proc. 21, 1350, 1989 and Yen T, Hanan S A, Johnson D E et al.; Ann. Thorac. Surg. 49, 932, 1990). Transplanting of rat hearts (n=5) immersed in a combination medium of UWS and perfluorocarbon was found to be successful at both 24 hours (100%) and 48 hours (4 out of the 5 animals) (Kuroda Y, Kawamura T, Tanioka T et al; Transplantation, 59, 699-701, 1995). The reason for these short time limits is that when removed hearts are exposed to the low temperature of 4xc2x0 C. or ischemic injury, their cell membranes are damaged to make tissue cell resuscitation impossible (Pegg D E; Organ Preservation Surg. Clin. North Am. 66, 617, 1986: Oz M C, Pinsky D J, Koga S et al.; Circulation 88, 291-297, 1993: and Heffner J E, Pepine J E; Rev. Pespir. Dis. 140, 531-554, 1989).
The technology for storing mammalian living tissues over prolonged periods before resuscitation have seen marked advances only in the area of single cells such as blood, sperm and ova. Efforts to develop practically feasible methods for the cold storage of living tissues which are aggregates of cells and organs which are composed of several tissues are also in progress but they have to meet the inexorable requirement that transplant be performed within 24 hours of storage (Kalayoglu M, Sollinger H W, Strarra R J et al.; Lancet 2, 617, 1988).
As regards the technology of organ preservation and resuscitation, trehalose (C12H22O11) is an interesting substance to mention. This is a nonreducing disacharide found widely in nature and it has been reported to have the ability to stabilize or protect the structure of cell membranes under various types of stress (Crowe J H, Crowe L M, Chapman D; Science 233, 701-703, 1984 and Wiemken A; Antinei Van Leeunwenhoek 58, 209-217, 1990). It was also reported that trehalose had the ability to protect cell membranes of the heart when it was exposed to the low temperature of 4xc2x0 C. or ischemic injury (Stringham J C, Southhard J H, Hegge J et al.; Transplantation 58, 287-294, 1992 and Hirata T, Fukuse T, Liu C J et al.; Surgery 115, 102-107, 1994).
According to reports of experiments with tardigrades under high hydrostatic pressure, trehalose increased 10-20 fold in an anhydrous state (Crowe J H, Crowe L M, Chapman D; Science 233, 701-703, 1984 and Crowe J H, Crowe L M, Chapman D, Aurell Wistorm; Biochemical Journal 242, 1-10, 1987). Tardigrades are multicellular organisms composed of ca. 40,000 cells including nerve cells.
The present inventor previously found that tardigrades in a desiccation state had the viability to withstand high hydrostatic pressures up to 600 MPa (Kunihiro Seki et al.; Nature Vol. 395, No. 6705, pp. 853-854, Oct. 29, 1998 and Japanese Patent Public Disclosure No. 289917/1999 which is incorporated herein by reference to the specification and the drawings). Tardigrades become xe2x80x9cdesiccatexe2x80x9d when they are in the xe2x80x9ctunxe2x80x9d state. The physiological mechanism behind their tun state has not been fully unravelled but it is at least clear that desiccated tardigrades have lost an extremely large amount of water in their body to become dehydrated.
As described above, the shortage of donors and the increasing time for which patients have to wait before surgery are two serious problems with organ transplants and a strong need exists to develop feasible techniques for preserving organs and later resuscitating them.
In the conventional storage of organs by refrigeration, the temperature of the organ is lowered so that its metabolism is suppressed to a level that maintains its viability. While the metabolism of the organ is suppressed by reducing temperature, water as a polar medium is a rich supply of ions which cause self-disintegration of cells, their death and necrosis over time. Hence, the longer the period of storage by refrigeration, the higher the frequency of the occurrence of serious thrombus formation and dysfunction. Organs cannot be stored cold for an indefinite period.
An object, therefore, of the invention is to provide a novel technique by which organs can be stored in vitro for a significantly increased number of days while preventing their cells and tissues from undergoing self-disintegration over time.
Another object of the invention is to provide a basic technique of such substantial utility that it can extend the duration of preservation of mammalian organs for use in transplanting into humans.
The present inventor found that the ability of desiccated tardigrades to withstand extreme environments in an inert medium could be applied to the purpose of preserving mammalian organs for an extended period. The organs preserved by the present invention can be later resuscitated for collecting viable nerves or stem cells. The resuscitated organs or the collected tissues can be used in transplants. For histopathological studies, it is quite significant that resuscitable biomaterials rather than necrotic specimens can be stored for a long period.
Animal tissue cells generally are not viable in the absence of water. One may readily imagine that organs of higher animals which are composed of heterogeneous tissues can never be resuscitated from a desiccation state. Techniques for preserving plants and various bacteria in a desiccation or dry state have already been developed but not a single experiment has been reported in which organs of higher animals were successfully resuscitated after storage in a desiccation or dry state.
To his surprise, the present inventor found that when extracted mammalian organs were deprived of much water under specified conditions and later stored at low temperature within an inert medium, they had apparent death of the same nature as experienced by tardigrades which remained viable in the tun state for a prolonged period.
Particularly surprising was that multi-cell and multi-tissue mammalian organs resuscitated from an extremely dehydrated state and that the resuscitated heart was found to beat. The cells of the resuscitated organ are believed to be in apparent death characterized by either an extreme drop in oxygen consumption (no greater than {fraction (1/1000)} of the normal level) or substantial arrest of oxygen consumption.
The method for preserving mammalian organs according to the first aspect of the invention comprises two steps, one of dehydrating an organ to remove water but leave intact an amount of water that permits later resuscitation and the other of immersing the organ in an inert medium and maintaining it at a chill temperature or below.
In a preferred case of the dehydration step, an organ having a physiologically normal water content is deprived of water in an amount of at least about 25% by weight of the total weight of the organ before dehydration such that water is left intact in an amount of at least from about 10 to about 20% by weight of the total content of water before dehydration. This step of dehydration is preferably followed by the step of immersing the organ in an inert medium and maintaining it at a chill temperature.
The preserved mammalian organ according to the second aspect of the invention is such that it is deprived of water in an amount of at least about 25% by weight of its physiologically normal, total weight while leaving water intact in an amount of at least from about 10 to about 20% by weight of the total water content in the organ which is then immersed in an inert medium and maintained at a chill temperature or below. Examples of such stored mammalian organs include heart, liver, kidneys, pancreas and lungs.
xe2x80x9cDepriving of water in an amount of at least about 25%xe2x80x9d means removing the body fluid in the vascular system, as well as the free water present in and between individual cells. xe2x80x9cLeaving water intact in an amount of at least from about 10 to about 20%xe2x80x9d shall be taken to mean that after removal of water, the organ still contains a sufficient amount of water to permit later resuscitation, inclusive of the bound water in the living tissue.
When free water is removed from the tissues and cells of the organ, biostructures such as biomembranes become less susceptible to the attack of substances, particularly metal ions, that can be activated in the aqueous phase. The biostructures are presumably protected by the surrounding water in a crystalline state called xe2x80x9cbound waterxe2x80x9d. As a result of the removal of the polar medium that degrades the living tissue, the tissues and cells of the organ become immune to degradation with time and the organ can be preserved in a significantly improved state. In this case, trehalose in the preserving solution can contribute to stabilizing the biostructure.
The freshness of stored organs is believed to depend primarily on the amount of free water and to prolong the preservation period, it is theoretically preferred to remove free water as much as possible. To maintain resuscitability, it is preferred to ensure that water is left intact in a range of amounts that enable the maintenance of bound water. Bound water may be defined as the water in which the state of hydration or crystallization can be observed, and free water as the water other than bound water.
Animal organs are generally understood to have a water content in the range from about 60 to about 80 mass%. A suitable state of dehydration will depend on the inherent water content of a specific kind of organs but all that is required by the present invention is that the organ to be preserved should be deprived of water in an amount of at least about 25% by mass of the total weight of the organ before dehydration so that the organ contains water in an amount of at least about 10 to about 20% by weight of the total water content before dehydration.
If the method of the invention is to be applied to preserving heart, it comprises three steps, the first for removing blood from the heart by flushing with physiological saline until the blood in the heart is replaced by the physiological saline, the second for depriving the flushed heart of water in an amount of from about 25 to about 60% by weight of the total weight of the heart before dehydration, and the third for immersing the dehydrated heart in an inert medium and maintaining it in the state of apparent death at a chill temperature between about 2 and about 4xc2x0 C. By removing about 25 to about 60% of water from the heart, water can be left intact in an amount of at least from about 10 to about 20% by weight of the total water content before dehydration.
Dehydration of organs can conveniently be accomplished by bringing the organ to be preserved into contact with a dehydrator and absorbing water from within the organ. Specifically, the washed and flushed organ is surrounded by the required amount of dehydrator and immersed in an inert, medium together with it. The immersed organ is gradually dehydrated in the inert medium until it suffers apparent death. Having been dehydrated to an extremely low water content, the organ stored cold in the inert medium can maintain chemical stability in all tissues including nerve tissue. In the experiments conducted by the present inventor, rat hearts could actually be preserved for as many as 10-20 days without suffering excessive damage to the nerve system.
In order to increase the resuscitation ratio and achieve further improvements in the state of preserved and resuscitated organs, a method of dehydration by withdrawing water from within the organ through channels in the vascular system may be employed in practicing the preservation method of the invention.
Specifically, this can be achieved by irrigation perfusion or flushing with a specified gas medium that can flow through arteries or veins connecting to capillaries in the organ until it displaces the water in the organ. The gas medium gets into capillaries from one end of the vascular system and creates a flow pressure that allows the gas medium to infuse all parts of the organ tissues at substantially the same rate; thereafter, the gas medium circulates through the capillaries (for example, from arterial to venous vessels) until it reaches the other end of the vascular system. As a result of this gas perfusion of the vascular system, the body fluid in the organ is pushed forward so that water is withdrawn from every one of the cells via capillaries. Gas perfusion can be effected with an irrigation apparatus for flushing physiological saline if the gas is pumped in instead of the physiological saline.
The gas to be supplied into the vascular system may be air or a gaseous mixture of O2 and CO2. Alternatively, inert gases may be used, as exemplified by N2, He, Ar, Ne, Kr and Xe.
In anatomy, vascular systems are classified into two groups, blood vessels and lymphatics. For the purposes of the invention, nutrition blood vessels through which water and nutrients are supplied to individual cells in the organ of interest can preferably be used as the xe2x80x9cvascular systemxe2x80x9d. In the case of the heart, the irrigation apparatus may be connected to inherent vascular vessels leading to the atria and ventricles so that flow pressure is applied indirectly to the coronary arteries and veins leading to the nutrition blood vessels in the heart. Besides the nutrition vessel system, organs such as the liver have a functional blood vessel system associated with portal vein circulation; in such organs, the functional blood vessel system may be substituted for the nutrition vessel system.
Liquids to be supplied to the vascular system include solvents that make use of osmotic pressure difference to displace water from cells, as exemplified by hypertonic liquids more concentrated than the body fluids in organs. These may be substituted by the inert medium to be described later, or alcohols.
Dehydration via the vascular system utilizes the water supply passages inherent in organs and can hence create a uniform dehydrated state at slow speed in the desired tissues or cells. Even in the case of mammalian, multi-cell and multi-tissue organs, transfer to a highly dehydrated state can be achieved smoothly without undue stress on the living tissue. The living tissue is usually placed under stress by 25 mass % or more dehydration; however, dehydration via the vascular system can bring organs to apparent death in an extremely stable state without causing ischemic injury or damaging the living tissue. Upon refilling with water, functional resuscitation occurs not only in the cells and tissues but also in the organs themselves.
Apparent death usually means biological apparent death. The term xe2x80x9capparent deathxe2x80x9d as intended by the invention should be taken to mean such a state that the external signs of xe2x80x9clifexe2x80x9d are lost as a result of enhanced dehydration but can be restored upon refilling with water. The term xe2x80x9cresuscitationxe2x80x9d as used herein means such a phenomenon that upon refilling with water, a dehydrated tissue or organ resumes recognizable electrophysiological reactions or biological life activities, respectively.
In the method of the invention, the step of dehydration using gases is preferably preceded by blood removal using physiological saline. Blood removal is effective in avoiding the problem of blood coagulation upon contact with the flushing gas.
The xe2x80x9corgan having a physiologically normal water contentxe2x80x9d is typically an organ as extracted from the living body. If the method of the invention includes the step of blood removal, this term can be taken to mean an organ whose blood has been replaced by physiological saline as a result of irrigation performed to effect blood removal. The degree of dehydration can be specified with reference to the weight of the xe2x80x9corgan having a physiologically normal water contentxe2x80x9d.
The physiological saline to be used in blood removal is a substitute body fluid having similar physiological activity to blood and typical examples are known Ringer""s solutions such as KH (Kreps-Henseleit) solution. Polysaccharides that will help stabilize the dehydrated biostructure may be dissolved in physiological salines of this class. A preferred polysaccharide having this ability is trehalose. Other biostructure stabilizing substances that can be dissolved in physiological saline include malic acid, mannitol, glycerol, and amino acids such as glycine betaine, proline and ectoine.
The inert medium to be used in the invention is a medium that is insoluble in water and oils and fluorocarbons that are liquid at the temperature for preservation are preferred, with a liquid perfluorocarbon being particularly preferred. If similar conditions are satisfied, other forms of inert medium may be used such as gas, sol and gel. Other inert media that are believed to be useful include mercury and silicone oil.
The heart, liver, kidneys, pancreas and lungs preserved by application of the present invention can be resuscitated by flushing their vascular system with body fluid or substituted body fluids that have been warmed to near body temperature, as exemplified by the physiological saline described above, artificial blood and/or natural blood. The resuscitated organs or tissues collected from them are believed to be transplantable to the human body. Nerve tissue and other living tissues and cells can be collected from the resuscitated organs and used for testing purposes as in a pharmacological test.
Another possible application of the invention is to organs of mammals except humans that can be used as heterologous transplants to the human body and by this application the invention provides an effective technique for preserving animal organs that are expected to find increasing demand in clinical settings to deal with the shortage of human donors. In particular, the invention is also useful for preserving mass-producible organs such as the heart, liver and pancreas from cultured pigs; thus, the invention will provide an effective preservation technique that can withstand transport for a long time, particularly air transport for a period longer than ten-odd hours.
The invention will also find utility in application to the storage of tissues and organs that have been reconstituted by cultivating stem cells having totipotency such as embryonic stem cells taken from blatocysts. The invention may find further applicability to the storage of nerve tissues in the brain and other organs.